Breast cancer is the second-leading cause of cancer deaths in women in the US. Despite initial treatment response, most patients progress to late-stage disease with metastases, likely due to resistant cancer stem cells (CSC) initiating tumor recurrence. CD44 and CD133 expression and increased ALDH1activity strongly correlate with breast cancer cells? tumor-initiating and metastatic potential, suggesting that CD44, CD133, and ALDH1 are markers for breast CSCs. Inhibition of mTOR by rapamycin is a viable approach to kill CSCs, which can be resistant to other types of chemotherapies. HylaPharm, LLC has developed a locally injectable nanoparticle formulation targeted toward breast CSCs through specific interactions of a hyaluronan (HA) and CD44. By conjugating rapamycin to HA (HA-rapa), rapamycin is delivered directly to primary tumors and draining lymphatics (where most metastases initially migrate). In this Phase I proposal, we will characterize breast cancer cell lines for CSC markers (CD44 and CD133 expression and high ALDH1 activity) and tumorigenicity, and then evaluate the ability of HA-rapa to specifically target CSCs in these cells lines and mouse models. Completion of our Phase I goals will provide the necessary basis for IND-enabling studies (Phase II proposal), with the goal of filing an IND application to the FDA.